José BA Custódio1,2, Mariana V Ribeiro1,2, Filomena SG Silva1,2, Marisa Machado3,4, M Céu Sousa3,4 1Center for Neuroscience and Cell Biology, 2Laboratory of Biochemistry, Faculty of Pharmacy, 3Center of Pharmaceutical Studies, 4Laboratory of Microbiology, Faculty of Pharmacy, University of Coimbra, Portugal Abstract: Essential oils can be used as antimicrobial, antioxidant, and anticarcinogenic agents or to preserve and give flavors to foods. The activity of phenolic-rich essential oils has been observed in fractions containing thymol and carvacrol which show synergistic effects with their precursor p-cymene. Their mode of action is related to several targets in the cell but specific mechanisms of activity and cytotoxic effects remain poorly characterized. Given the importance of mitochondria for cellular functions and their critical role in a vast number of diseases, this work evaluated the effects of p-cymene on mitochondrial functions. It was observed that p-cymene did not change the oxygen consumption by respiratory chain (state 2 respiration). However, p-cymene decreased the mitochondrial membrane potential (Δψ), depressed the rate of ADP phosphorylation (state 3), and stimulated the oxygen consumption after phosphorylation of ADP (state 4). The respiratory control ratio (state 3/state 4) was decreased as a consequence of the inhibition of state 3 and stimulation of state 4 respiration but the ADP/O index remained unaltered as well as the mitochondrial Ca2+ fluxes. Moreover, p-cymene did not induce mitochondrial membrane disruption but depressed the Δψ, and the stimulatory effect observed on state 4, similar to the effect observed on state 2 respiration plus ATP, was inhibited by oligomycin. These effects suggest that p-cymene allows a proton leak through the Fo fraction of the phosphorylative system, changing the mitochondrial proton motive force and ATP synthesis capacity. Therefore, these data suggest mitochondria as a target for p-cymene toxicity action mechanisms. Keywords: antimicrobial, proton leak, ATP synthase, p-cymene, essential oils, food additive, mitochondrial respiration, uncoupling